The ultimate objective of this research project is to identify the molecular mechanism (and/or key mediators) by which osteoclastic resorption is regulated. In this regard, recent studies showed that targeted over-expression of a structurally unique osteoclastic protein-tyrosine phosphatase (PTP-oc) in osteoclastic cells led to a large increase in bone resorption and a marked decrease in bone mass in male but not female transgenic mice, compared to corresponding wild-type littermates. Very recent preliminary studies discovered two novel molecular mechanisms of PTP-oc to regulate the overall osteoclast activity: 1) the EphA4 signaling may function as a negative regulatory mechanism of osteoclastic resorption and PTP-oc stimulates osteoclast differentiation and activity through relieving the inhibitory actions of the EphA4 signaling via dephosphorylation of EphA4;and 2) the PTP-oc signaling increases estrogen receptor (ER) 1 signaling through an upregulation of the c-Src-dependent phosphorylation of ER1 in osteoclasts, which in turn suppresses bone resorption in adult female but not male PTP-oc transgenic mice. This proposal has three Specific Objectives to investigate these two novel mechanisms of PTP-oc. The first Objective is to demonstrate that the EphA4 signaling is negative regulator of osteoclasts through differential regulation of the various Rho GTPases and through suppression of the Erk1/2-c-Fos-NFAT1c cascade. This will be accomplished through evaluation of the bone phenotype of EphA4-deficient mice in vivo and to determine the effects of deficient EphA4 expression in osteoclast precursors of EphA4 null mice or transgenic over-expression of EphA4 in wild-type precursors on the formation, fusion, adhesion, migration, actin cytoskeleton re-organization, and resorption activity of osteoclasts in vitro. Effects of deficient EphA4 signaling in EphA4 null osteoclasts on the activation states of the various Rho GTPases and the Erk1/2-c-Fos-NFATc1 signaling cascade are also determined. The second Objective is to determine whether PTP-oc enhances by osteoclastic resorption, in part, through suppressing the inhibitory actions of the EphA4 on osteoclasts through direct dephosphorylation of EphA4. This is achieved by confirming that EphA4 is a cellular substrate of PTP-oc, and by demonstrating that over-expression of WT-PTP-oc, but not PD-PTP-oc, would inhibit the suppressive effects of the EphA4 signaling on osteoclast differentiation and activity. It is also tested by showing that the hemotopoietic stem cell-based marrow transplantation with WT- EphA4, but not the mutant lacking the key phosphotyrosine residues, would reverse the osteoporotic phenotype of EphA4 null mice. The third Objective is to determine whether the PTP-oc signaling increases estrogen receptor (ER) 1 signaling through the c-Src-dependent phosphorylation of ER1 in osteoclasts, which in turn suppresses bone resorption in adult female but not male PTP-oc transgenic mice. This is achieved by a) comparing the bone phenotype and the ER1 signaling in osteoclasts of pre-pubertal male and female PTP-oc transgenic mice, 2) evaluating the effects of ovariectomy and estrogen replacement on the osteoclastic phenotype of adult female PTP-oc transgenic mice, and 3) evaluating the effects of PTP-oc over-expression in osteoclasts of ER1 null female mice. Our work should provide important insights into the molecular mechanism of the osteoclast activation and/or the role of a unique osteoclastic enzyme (PTP-oc) in the regulation of two novel signaling mechanisms of osteoclastic resorption. Potential clinical relevance is that aberration in PTP-oc function could be involved in some patients with excess bone resorption and, as such, PTP-oc could be a target for pharmacogenomic treatments of osteoporosis and related bone-wasting diseases. PUBLIC HEALTH RELEVANCE: Bone diseases, including osteoporosis and related bone-wasting diseases, are common in the VA patient population. The increase in incidence of osteoporosis and related diseases is particularly high in female veterans. Identification of the etiology and molecular mechanism of such bone loss and effective therapies to overcome and/or even prevent osteoporosis would significantly reduce the morbidity with aging and improve the quality of life. A reduction in hip fractures, which can be a particular debilitating symptom of osteoporosis, would be desirable with respect to reduction of medical costs and human suffering. Increased understanding of the cause and mechanisms of osteoclastic resorption, which is the major objective of this project, should provide a foundation of knowledge to improve our understanding of the cause of bone-wasting diseases. This project may allow disclose novel targets for development of more effective anti-resorptive therapies for osteoporosis and related disease. Thus, this project is highly relevant to the VA patient care mission.